Synthesis and Characterization

Compound 2: 6-S-[Methyl 5-acetamido-7, 8, 9-tri-O- acetyl-4-azido-3, 4, 5-trideoxy -D-

glycero-α-D-galacto-non-2-ulopyranosyl) onate]-1-chloro-hexane.

To a stirring solution of compound 166 (0.50 g, 0.94 mmol) in DMF (1.0 ml), DEA (0.95 ml, 9.4 mmol) was added. The reaction mixture was stirred for 10 min at rt. 6-Chlorohexyl 4- methylbenzenesulfonate (0.35 g, 1.1 mmol) was added and the reaction stirred for additional 12 h. The reaction mixture was quenched using brine solution (0.20 L, 1x) and extracted with ethyl acetate (0.050 L, 3x). Organic layers were combined and washed with HCl solution (0.05 ml, 1M, 1X), dried over Na2SO4 and concentrated in vacuo. The title compound was purified using column chromatography with hexane:acetone (3:1) as eluent to yield a yellow oil (0.46 mg, 80% yield). 1H NMR (400 MHz, CDCl3) δ 5.57 (d, J = 8 Hz, 1H), 5.39-5.36 (m, 1H), 5.31 (d, J = 7.6 Hz, 1H), 4.34-4.30 (m, 1H), 4.25-4.19 (m, 1H), 4.11 (d, J = 10.8 Hz, 1H), 4.06-4.02 (m, 1H), 3.83 (s, 3H), 3.56 (vt, J = 6.4, 13.2 Hz, 2H), 3.33-3.25 (m, 1H), 2.82-2.73 (m, 2H), 2.59-2.53 (m, 1H), 2.18 (d, J = 3.2 Hz, 6H), 2.06 (s, 3H), 2.01 (s, 3H), 1.81-1.73 (m, 3H), 1.55-1.39 (m, 6H). 13_{C-NMR (100 MHz, CDCl} 3) δ 170.8, 170.7, 170.6, 170.0, 168.3, 82.9, 72.8, 68.3, 68.0, 62.0, 58.0, 52.3, 45.0, 38.2, 32.4, 29.1, 28.7, 28.0, 26.3, 23.4, 21.1, 21.0, 20.7. HRMS (ESI): Calculated for: C24H37ClN4O10SH, 608.1919; found, 609.1975 (M+H).

59 Compound 3a: 6-S-[Methyl 5-acetamido-7, 8, 9-tri-O- acetyl-3, 4, 5-trideoxy -4-(N-tert-

butyloxycarbonyl)-amino-D-glycero-α-D-galacto-non-2-ulopyranosyl)onate]-1-azido- hexane.

To a stirring solution of compound 2 (0.20 g, 0.33 mmol) in a mixture of THF: H2O (5.0 ml, 1:1), PPh3 (0.10 g, 0.39 mmol) was added and the reaction was heated at 40 °C for 12 h. The solvent was removed in vacuo and the residue purified by column chromatography to obtain a clear oil. This compound was directly taken to the next step without further purification. To a stirring solution of the crude product (0.18 g, 0.31 mmol) in THF (0.010 L) and TEA (31 mg, 0.31 mmol), di-t-butyl carbonate anhydride (0.10 g, 0.47 mmol) was added. Reaction mixture

was stirred for 12h at rt. The white solid was filtered and the residue was concentrated, dissolved in DCM (25 ml) and washed with HCl (25 ml, 1M, 1x). The organic layer was dried over Na2SO4 and concentrated in vacuo. Product was purified via column chromatography with hexane: acetone (5:1) to yield clear yellow oil (0.13 g, 60% yield for two steps). 1_{H NMR (400} MHz, CDCl3) δ 5.46 (s, 1H), 5.33 (s, 2H), 4.75 (d, J = 8.8 Hz, 1H), 4.28 (d, J = 12 Hz, 1H), 4.09 (d, J = 8.8 Hz, 1H), 3.88 (d, J = 10.4 Hz, 1H), 3.79-3.73 (m, 5H), 3.52 (t, J = 6.4, 3H), 2.72 (d, J

= 11.6 Hz, 2H), 2.54-2.48 (m, 1H), 2.15 (s, 3H), 2.11 (s, 3H), S-4 2.03 (s, 3H), 1.88 (s, 4H), 1.77-1.71 (m, 4H), 1.38 (s, 19H). 13CNMR (100 MHz, CDCl3) δ 171.0, 170.7, 170.0 169.9, 168.7, 156.0, 83.4, 80.0, 68.5, 67.5, 62.2, 52.9, 50.2, 50.0, 45.0, 39.2, 32.4, 29.1, 28.7, 28.3, 26.4, 23.2, 21.2, 20.8, 20.7. HRMS (ESI): Calculated for C29H47ClN2O12S: 682.2538; Found: 683.2613 (M+H).

60 To a stirring solution of the chloro derivative (71 mg, 0.10 mmol) in DMF (1.0 ml) NaN3 (65 mg, 1.04 mmol) was added and the reaction was stirred at 60 °C. After 12 hr, the reaction mixture was quenched with DI water (25 ml) and extracted with DCM (25 ml, 3x). The organic layers were combined, washed with brine (25 ml, 1x), dried over Na2SO4 and concentrated in vacuo, to yield a clear oil (64 mg, 90% yield). 1H NMR (400 MHz, CDCl3) δ 5.47 (d, J = 12 Hz, 1H), 5.33 (s, 2H), 4.75 (d, J = 8 Hz, 1H), 4.29 (d, J = 12 Hz, 1H), 4.09 (d, J = 8 Hz, 1H), 3.87 (m,

1H), 3.79 (s, 3H), 3.60 (m, 1H), 3.52 (vt, J = 12, 8.0 Hz, 2H), 2.73 (d, J = 12 Hz, 2H), 2.54-2.48

(m, 1H), 2.15 (s, 3H), 2.11 (s, 3H), 2.03 (s, 3H), 1.88 (s, 3H), 1.77-1.74 (m, 3H), 1.5-1.40 (m, 2H), 1.38 (s, 14H). 13C NMR (100 MHz, CDCl3) δ 171.0, 170.7, 170.1, 170.0, 168.7, 156.0, 83.4, 80.0, 68.5, 67.4, 62.2, 52.9, 50.2, 50.0, 45.0 39.2, 32.4, 28.3, 26.4, 23.2, 21.2, 20.84, 20.80. HRMS (ESI): Calculated for C29H47N5O12S: 689.2942; Found: 690.3004 (M+H).

Compound 3b: 6-S-[Methyl 5-acetamido-7, 8, 9-tri-O- acetyl-3, 4, 5-trideoxy-4-(bis-N, N'- tert-butyloxycarbonyl)-guanidino-D-glycero-α-D-galacto-non-2-ulopyranosyl)onate]-

hexane

To a stirring solution of compound 2 (0.11 g, 0.20 mmol) in a mixture of THF and H2O (2.0 ml, 1:1), PPh3 (62 mg, 0.24 mmol) was added and the reaction was heated at 40 °C for 12 h. The solvent was removed in vacuo and purified by column chromatography using DCM:MeOH (10:1). To this compound (0.10 g, 0.16 mmol) in DCM (0.01 L), TEA (0.3 ml, 1.6 mmol) was added and the reaction was stirred for 10 min at rt. Then 1,3-bis(tert-butoxy-carbonyl)-2-methyl-

61 2-thiopseudourea (55 mg, 0.19 mmol) and HgCl2 (51 mg, 0.19 mmol) were added and the reaction was stirred for 12 h. Next, the reaction mixture was washed with H2O (25 ml, 1x) and brine (25 ml, 1X). The organic layers were separated, dried over Na2SO4 and concentrated in vacuo. Product was purified using column chromatography with hexane:acetone (3:1 ratio) to yield a clear oil (0.12 g, 80% yield). 1H NMR (400 MHz, CDCl3) δ 11.30 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 6.05 (d, J = 8.0 Hz, 1H), 5.37-5.29 (m, 2H), 4.36-4.33 (m, 1H), 4.06 (s, 3H), 3.54 (t, J = 12, 4.0 Hz, 2H), 2.82-2.74 (m, 2H), 2.56-2.53 (m, 1H), 2.17 (s, 3H), 2.14 (s, 3H), 2.04 (s,

3H), 1.83 (s, 4H), 1.48 (s, 24H). 13C NMR (100 MHz, CDCl3) δ 170.8, 170.7, 170.2, 170.1, 168.8, 163.0, 156.8, 152.7, 83.8, 83.3, 79.5, 75.4, 69.0, 67.8, 62.4, 52.9, 50.5, 49.7, 45.0, 38.8, 32.4, 29.1, 28.7, 28.3, 28.0, 27.9, 26.4, 23.0, 21.2, 21.0, 20.8. HRMS (ESI): Calculated for C35H57ClN4O14S: 824.3281; Found: 825.3348 (M+H).

To a stirring solution of the chloro compound (47 mg, 0.06 mmol) in DMF (1.0 ml), NaN3 (39 mg, 0.60 mmol) was added and stirred at 60 oC. After 12 h, the reaction mixture was quenched with H2O and extracted with DCM (25 ml, 3x). The organic layers were collected and washed with brine (25 ml, 1x), dried over Na2SO4 and concentrated in vacuo, to yield a clear oil (40 mg, 85% yield). 1_{H NMR (400 MHz, CDCl} 3) δ 11.31 (s, 1H), 8.40 (d, J = 7.0 Hz, 1H), 6.03 (d, J = 8.7 Hz, 1H), 5.33 (t, J = 15.6 Hz, 4H), 4.35 (d, J = 12.4 Hz, 2H), 4.14 – 3.94 (m, 4H), 3.91 – 3.69 (m, 7H), 3.27 (t, J = 6.8 Hz, 4H), 2.99 – 2.47 (m, 9H), 2.17 (s, 5H), 2.15 (s, 5H), 2.05 (s, 6H), 1.84 (s, 5H), 1.60 (d, J = 6.3 Hz, 6H), 1.49 (s, 22H), 1.39 (s, 10H), 1.26 (s, 8H). 13C NMR (100 MHz, CDCl3) δ 170.8, 170.6, 170.2, 170.1, 168.8, 162.9, 156.8, 152.7, 83.8, 83.3, 79.5, 77.3, 77.0, 76.7, 75.4, 69.0, 67.8, 62.4, 52.9, 51.3, 50.4, 49.7, 38.8, 29.7, 29.1, 28.7, 28.7, 28.3, 28.0, 26.2, 23.0, 21.2, 21.0, 20.8. HRMS (ESI): Calculated for C35H57N7O14S: 831.3684; Found: 832.3695 (M+H).

62 Compound 4: tert-Butyl(7-((3,5-bis(prop-2-yn-1-ylcarbamoyl)phenyl)amino)-7-oxoheptyl)

carbamate.

To a stirring solution of the known spacer (64 mg, 0.26 mmol) in THF (5.0 ml), CDMT (91 mg, 0.52 mmol) and NMM (52 mg, 0.52 mmol) was added at 0 oC. In a separate flask, the known dimeric scaffold 2 was dissolved in THF (5 ml) with NMM (52 mg, 0.52 mmol) at 0 oC and added to the activated acid and stirred for 12 h. The reaction mixture was quenched with H2O, extracted with EtOAc (10 ml, 3x), dried over Na2SO4 and concentrated in vacuo. The compound was purified using flask chromatography with hexane: acetone (3:1) to yield a white solid (96 mg, 80% yield). 1H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 8.27 (s, 2H), 8.04 (s, 1H), 7.75 (s, 2H), 4.80 (s, 1H), 4.17 (s, 4H), 3.00 (d, J = 6.1 Hz, 2H), 2.25 (t, J = 37.5 Hz, 5H), 1.42 (s,

15H). 13C NMR (100 MHz, CDCl3) δ 173.00, 166.66, 159.37, 139.39, 134.58, 121.77, 79.42, 77.36, 77.05, 76.73, 71.69, 56.03, 37.05, 31.58, 30.94, 29.79, 28.60, 28.45, 26.29, 25.27, 22.64, 14.11. HRMS (ESI) Calculated for C26H34N4O5: 482.2529; Found: 505.2404 (M+Na).

63 To a stirring solution of 3a (40 mg, 0.06 mmol) in THF/ H2O (1.0 ml, 1:1), 4 (9.6 mg, 0.02 mmol) was added. CuSO4 (10 mg, 0.04 mmol) was added along with sodium L-ascorbate (7.9 mg, 0.04 mmol) and the reaction was stirred at rt for 12 h. Solvent was removed in vacuo and product was purified using flash column chromatography with DCM:MeOH (10:1) to yield 5a (33 mg, 60% yield). 1H-NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.35 (s, 3H), 8.07 (s, 3H), 6.08 (s, 2H), 5.53 (d, J = 8 Hz, 2H), 5.36-5.31 (m, 5H), 4.70 (s, 5H), 4.32-4.29 (m, 6H), 4.07 (dd, J = 4, 12 Hz, 2H), 3.99-3.92 (m, 2H), 3.78-3.75 (m, 3H), 3.71 (s, 6H), 3.55 (d, J = 8 Hz, 2H), 3.10 (d, J = 4 Hz, 2H), 2.72-2.65 (m, 6H), 2.55-2.51 (m, 2H), 2.38 (s, 2H), 2.19 (m, 3H), 2.13 ( s, 6H), 2.02 (s, 6H), 1.98 (s, 6H), 1.93 (s, 6H), 1.45 (s, 12H), 1.36 (s, 27H), 1.27 (s, 12H). 13_C NMR (100 MHz, CDCl3) δ 210.8, 172.3, 171.3, 170.7, 170.1, 170.0, 168.8, 166.6, 156.3, 156.1, 139.7, 135.0, 134.9, 123.1, 121.5, 121.44, 121.4, 121.3, 83.5, 79.7, 79.13, 79.11, 77.9, 77.6, 77.5, 77.2, 76.6, 76.5, 74.4, 69.5, 68.4, 68.2, 68.0, 62.4, 53.8, 52.9, 51.0, 50.3, 50.2, 50.15, 50.08, 50.00, 40.5, 40.4, 38.8, 37.2, 37.1, 35.5, 35.4, 31.9, 31.8, 29.9, 29.8, 29.78, 29.7, 29.6, 29.5, 29.4, 29.3, 28.8, 28.7, 28.73, 28.69, 28.65, 28.62, 28.5, 28.4, 28.3, 28.2, 28.18, 28.14, 27,3, 27.2, 26.4, 25.9, 25.6, 25.5, 25.4, 25.3, 25.2, 23.3, 23.2, 22.7, 22.6, 21.1, 21.0, 20.8, 20.7, 14.1. HRMS (ESI) Calculated for C84H128N14O29S2: 1860.8413; Found: 1923.6098 (M+ 3Na).

64 To a stirring solution of compound 3b (26 mg, 0.04 mmol) in THF: H2O (1.0 ml, 1:1), 4 (8.6 mg, 0.017 mmol) was added. CuSO4 (7.5 mg, 0.38 mmol) was added with sodium L- ascorbate (6.7 mg, 0.28) and the reaction was stirred for 12h at rt. Solvent was removed in vacuo and the product was purified using flash column chromatography with DCM:MeOH (10:1) to yield compound 5b (84 mg, 65% yield). 1H NMR (400 MHz, CDCl3) δ 11.30 (s, 2H), 8.50 (d, J = 27.2 Hz, 3H), 8.38 – 8.19 (m, 2H), 7.86 – 7.59 (m, 6H), 6.25 (s, 2H), 5.33 (m, 5H), 4.72 (m, 6H), 4.34 (m, 7H), 4.21 – 4.01 (m, 9H), 3.88 – 3.69 (m, 10H), 3.11 (d, J = 6.1 Hz, 1H), 2.87 – 2.61 (m, 5H), 2.58 – 2.44 (m, 2H), 2.38-1.87 (m, J = 7.3 Hz, 22H), 1.57 – 1.14 (m, 67H), . 13C NMR (101 MHz, CDCl3) δ 170.9, 170.7, 170.2, 168.7, 166.3, 162.9, 156.8, 152.5, 144.6, 134.9, 122.9, 121.3, 114.1, 83.5, 77.3, 77.2, 77.0, 76.7, 75.0, 68.8, 67.8, 62.5, 56.1, 53.8, 52.9, 50.2, 35.5, 31.9, 29.8, 29.7, 29.4, 29.3, 28.6, 28.4, 28.2, 27.9, 27.5, 25.8, 23.0, 22.7, 21.2, 20.9, 20.8, 14.1. HRMS (ESI) Calculated for C96H148N18O33S2: 2144.9898; Found: 2145.9985 (M+H). Compound SC1.

65 To a stirring solution of compound 3a (10 mg, 16 µmol) in MeOH(1.0 ml), NaOMe (5.4 M, 0.25 ml) was added. The reaction was stirred for 1 h, neutralized with Dowex H+ resin until the pH was 7. The resin was filtered, solvent was removed in vacuo and the residue was re- dissolved in DCM: TFA (1.0 ml, 1:1) and stirred for 1 h and solvent removed in vacuo. The residue was dissolved in EtOH with calaytic amount of Pd(OH)2 and stirred for 8 h. Reaction mixture was filtered and solvent removed, re-dissolved in MeOH (1.0 ml), NaOH (10 mM, 1.0 ml) was added and stirred for 1 h. Dowex H+ resin was used to neutralize to pH 7 and solvent was removed and compounds were purified with Bio-Gel P-2 Gel with DI water as solvent (5.0 mg, 75% yield). 1H NMR (400 MHz, D2O) δ 3.72 (t, J = 11.4 Hz, 2H), 3.62 – 3.32 (m, 5H), 3.22 (s, 1H), 2.87 (t, J = 7.6 Hz, 1H), 2.75 – 2.41 (m, 4H), 1.91 (d, J = 13.9 Hz, 3H), 1.52 (m,

4H), 1.28 (s, 4H). HRMS (ESI) Calculated for C17H33N3O7S: 423.2039; Found: 422.1962 (M-1, negative ion).

Compound SC2.

SC2 was synthesized in a manner from 3b (5.0 mg, 16 µmol) similar to that of Sc1 and the product was purified using Bio-Gel P-2 Gel with DI water as solvent to give pure SC2 ( 1.9 mg, 70 %). 1H NMR (400 MHz, D2O) δ 4.25-4.08 (m, 3H), 3.99 (m, 3H), 3.58-3.55 (m , 1H), 3.36-3.34 (m, 1H), 3.12-3.07 (m, J = 8.2 Hz, 2H), 2.95-2.78 (m, 1H), 2.44-2.35 (m, 3H), 2.15 (s,

1H), 2.03 (s, 1H), 1.87 (s, 5H), 1.69-1.54 (m, 3H). HRMS (ESI) Calculated for C18H35N5O7S 465.2257; Found: 464.1987 (M-1, negative ion).

66 Compound SC3.

To a stirring solution of compound 5a (4.0 mg, 2.4 µmol) in MeOH (1.0 ml), NaOMe (5.4 M, 0.25 ml) was added. The reaction was stirred for 1 h, neutralized with Dowex H+ resin. The resin was filtered, solvent was removed in vacuo and residue was re-dissolved in DCM/TFA (1.0 ml, 1:1) and stirred for 1 h. Solvent was removed in vacuo, re-dissolved in MeOH (1.0 ml) and NaOH (10 mM, 1.0 ml) was added and stirred for 1 h. Dowex H+ resin was used to neutralized to pH 7 and solvent was removed. The product was purified with Bio-Gel P-2 Gel with DI water as solvent (2.2 mg, 80 % yield ). 1H NMR (400 MHz, D2O) δ 7.88 – 7.69 (m, 5H), 4.21 (q, J = 6.6 Hz, 2H), 4.06 – 3.90 (m, 1H), 3.76 – 3.44 (m, 11H), 3.30 – 3.14 (m, 2H), 2.90 –

2.83 (m, 2H), 2.80 – 2.70 (m, 2H), 2.62 – 2.47 (m, 3H), 2.45 – 2.33 (m, 2H), 2.32 – 2.21 (m, 2H), 1.92 (s, 6H), 1.77 (q, J = 12.9 Hz, 2H), 1.72 – 1.61 (m, 4H), 1.57 – 1.45 (m, 4H), 1.43 – 0.91 (m,

20H). HRMS (ESI) Calculated for C55H88N14O17S2: 1280.5893; Found: 641.3013 (2M+H). Compound SC4

67 SC4 was synthesized in a manner similar to SC3 using 5b (5.1 mg, 2.4 µmol) to yield a white solid (2.3 mg, 70% yield). 1_{H NMR (400 MHz, D}

2O) δ 7.99 – 7.73 (m, 5H), 4.28 (d, J = 6.2 Hz, 1H), 4.05 (s, 1H), 3.83 (m, 3H), 3.65 (m, 10H), 3.48 (dd, J = 25.8, 14.8 Hz, 10H), 2.68 –

2.43 (m, 5H), 2.46 – 2.23 (m, 4H), 1.87 (s, 6H), 1.81 – 1.46 (m, 10H), 1.42 – 0.98 (m, 18H). HRMS (ESI) Calculated for C57H92N18O17S2: 1364.6329; Found 1365.6420 (M+H).

Compound 7: 5-Acetylamino-4- N-tert-butyloxycarbonyl-6- (1,2,3-triacetoxy-propyl) -5,6- dihydro-4H-pyran-2-carboxylic acid methyl ester.

To a solution of compound 665b, 65c (1.5 g, 3.3 mmol) in EtOH (25 ml), Lindlar catalyst (0.15 g, 0.10 equivalent) was added. H2 gas was bubbled to the solution and the reaction was stirred at rt for 12 h. After filtering using celite, the filtrate was collected and solvent removed in vacuo to give a white product (1.4 g, quantitative). To this compound (0.53 g, 1.2 mmol) in THF (20 ml), Et3N (1.6 ml, 1.5 mmol) was added. The solution was stirred rt for 30 min and Boc2O (0.54 g, 2.5 mmol) was added and reaction stirred for 12h at rt. Upon completion, THF was removed in vacuo. The residue was washed using HCl (1M, 25 ml) and extracted by DCM (30

68 ml, 3x), the organic phases were combined and dried over Na2SO4. DCM was removed in vacuo and the reaction mixture was purified using column chromatography with hexane: acetone (3:1) as eluent to give a white product. (0.56 g, 86% yield). 1H NMR (400 MHz, CDCl3) δ 6.46 (d, J = 9.0 Hz, 1H), 5.94 (s, 1H), 5.43 (s, 1H), 5.27 (s, 1H), 4.63 (d, J = 12.4 Hz, 1H), 4.44 (d, J = 10.0 Hz, 1H), 4.35 (d, J = 8.9 Hz, 1H), 4.15 (dd, J = 12.3, 7.1 Hz, 1H), 3.95 (d, J = 9.2 Hz, 1H), 3.77 (s, 3H), 2.14 (s, 9H), 2.10 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.9, 170.6, 170.3, 170.0, 161.8, 156.2, 144.6, 111.0, 80.2, 71.4, 67.8, 62.2, 60.4, 52.4, 50.1, 47.3, 28.2, 23.1, 21.0, 20.9, 20.8, 20.7, 14.2. HRMS. Calculated for C23H34N2O12: 530.2112; Found: 531.2181 (M+H)

Compound 8: 5-Acetylamino-4- N-tert-butyloxycarbonyl-6-[(2,2-dimethyl-[1,3]dioxolan-4-

yl] 5,6-dihydro-4H-pyran-2-carboxylic acid methyl ester.

To a solution of compound 7 (0.56 g, 1.1 mmol) in MeOH (0.010 L), NaOMe (0.05 eq) was added. The solution was stirred at rt for 5 h. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was neutralized by H+ resin, the suspension was filtered. The liquid phase was collected and dried in vacuo to give a colorless compound. To a solution of this compound (0.42 g, 1.1 mmol) in dry acetone (0.010 L), H+ _{resin was added to} adjust the pH to 4. The solution was stirred at rt for 12 h. The suspension was filtered, acetone was removed in vacuo, the residue was washed by saturated NaHCO3, extracted by DCM (3x 0.020 mL), the organic phases were combined and dried over Na2SO4, DCM was removed in

69 vacuo and the reaction mixture was purified by flash column chromatography using hexane: acetone (4:1) to give the 3. (0.41 mg, 88%). 1H NMR (400 MHz, CDCl3) δ 6.65 (d, J = 6.4 Hz, 1H), 5.79 (s, 1H), 5.08 (d, J = 4.2 Hz, 1H), 4.82 (d, J = 9.0 Hz, 1H), 4.59 (t, J = 9.4 Hz, 1H),

4.38 (dd, J = 13.5, 5.3 Hz, 1H), 4.26 – 4.06 (m, 2H), 4.01 (d, J = 10.6 Hz, 1H), 3.91 (td, J = 10.1,

6.7 Hz, 1H), 3.76 (s, 3H), 3.50 (dd, J = 8.3, 4.3 Hz, 1H), 2.03 (s, 3H), 1.44 (s, 9H), 1.40 (s, 3H),

1.36 (s, 3H). 13C NMR (400 MHz, CDCl3) δ 173.9, 162.0, 157.2, 146.3, 109.2, 107.8, 81.0, 78.3, 77.3, 77.0, 76.7, 74.0, 69.7, 67.3, 52.4, 52.1, 48.7, 28.2, 27.1, 25.3, 23.0. HRMS (ESI) Calculated for C20H32N2O9: 444.2108; Found: 445.2180 (M+H).

Compound 9: 5-Acetylamino-4-N-tert-butyloxycarbonyl-6- [(2,2-dimethyl-[1,3]dioxolan-4-

yl)- (4-nitro-phenoxycarbonyloxy)-methyl]-5,6-dihydro-4H-pyran-2-carboxylic acid methyl ester.

To a solution of compound 3 (35 mg, 0.079 mmol) in pyridine (0.010 L), DMAP (19 mg, 0.16 mmol) was added. The solution was stirred at rt for 30 min and 4-nitrophenylchloroformate (32 mg, 0.16 mmol) was added. The reaction was stirred at rt for 16 h. The reaction mixture was washed by HCl (1 M, 0.025 L) and extracted with DCM (3 x 0.020 L), the organic phases were combined and dried over Na2SO4. DCM was removed in vacuo and the product was purified by column chromatography using hexane: acetone (3:1) to give 4. (39 mg, 80% yield). 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 8.9 Hz, 2H), 7.50 (d, J = 8.9 Hz, 2H), 5.97 (d, J = 9.6 Hz, 1H), 5.90 (s, 1H), 5.31 (t, J = 5.7 Hz, 1H), 4.81 (d, J = 9.6 Hz, 1H), 4.54 (t, J = 9.7 Hz, 1H), 4.47 –

70 4.29 (m, 2H), 4.24 (dd, J = 8.9, 5.7 Hz, 2H), 4.17 – 4.05 (m, 1H), 3.79 (s, 3H), 1.95 (s, 3H), 1.42

(s, 9H), 1.41 (s, 3H), 1.38 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 171.3, 161.6, 156.4, 155.7, 152.5, 145.6, 144.8, 125.2, 122.3, 110.5, 108.9, 80.6, 77.3, 77.0, 76.7, 75.1, 74.2, 65.5, 52.5, 49.5, 48.1, 28.2, 26.4, 25.5, 23.2. HRMS (ESI) Calculated for C27H35N3O13: 609.2170; Found: 632.2057 (M+Na).

Compound 10a: 5-Acetylamino-4-N-tert-butyloxycarbonyl-6-[(6-azido-hexylcarbamoyloxy)

-(2,2-dimethyl-[1,3]dioxolan-4-yl)-methyl]-5,6-dihydro-4H-pyran-2-carboxylic acid methyl ester. O N H N₃ O NHBoc COOMe AcHN O O O

To a solution of 6-azidohex-1-amine (19 mg, 0.13 mmol) in CH3CN (8.0 ml), Et3N (20 mg, 0.19 mmol) was added. The solution was stirred at rt for 30 min. 9 (39 mg, 0.064 mmol) in CH3CN (2.0 ml) was added. The reaction was stirred at rt for 3 h. The progress of the reaction was monitored by TLC. Upon completion, CH3CN was removed in vacuo and the reaction mixture was washed by HCl (1 M, 0.025 L), extracted by DCM (3 x 0.020 L), the organic phases were combined and dried over Na2SO4. DCM was removed in vacuo and the product was purified by column chromatography using hexane: EtOAc (1:1) to give 5. (35 mg, 89%). 1_H NMR (400 MHz, CDCl3) δ 6.15 (d, J = 9.4 Hz, 1H), 5.90 (s, 1H), 5.24 (d, J = 3.7 Hz, 1H), 5.01 – 4.83 (m, 2H), 4.51 (t, J = 8.9 Hz, 1H), 4.33 (dd, J = 13.6, 7.8 Hz, 2H), 4.19 – 4.04 (m, 2H),

4.04 – 3.92 (m, 2H), 3.76 (s, 3H), 3.73(s, 1H) 3.24 (t, J = 6.9 Hz, 2H), 3.11 (dt, J = 20.7, 6.6 Hz,

71 1.23(s, 3H). 13C NMR (101 MHz, CDCl3) δ 170.8, 162.1, 156.2, 155.5, 144.4, 111.4, 108.9, 80.1, 74.9, 69.7, 65.9, 52.4, 51.3, 50.0, 47.6, 41.1, 29.6, 28.7, 28.3, 26.4, 26.3, 26.3, 25.4, 23.2. HRMS (ESI) Calculated for C27H44N6O10: 612.3119; Found: 635.3006 (M+Na).

Compound 10b: 5-Acetylamino-4-[2,3-bis(tert-butoxycarbonyl)guanidine]-6-[(6-azido- hexyl carbamoyloxy)-2,3 dihydroxy propyl]-5,6-dihydro-4H-pyran-2-carboxylic acid methyl ester. O N H N3 O NH COOMe AcHN HO HO O NHBoc BocN

To a solution of compound 10a (40 mg, 0.084 mmol) in THF (3.0 ml), TFA (3.0 ml) was added, the reaction was stirred at rt for 1 h. THF was removed in vacuo, Et3N (26 µl, 0.25 mmol) was added. The solution was stirred at rt for 30 min. HgCl2 (27 mg, 0.10 mmol) and 1,3-Bis(tert- butoxycarbonyl)-2-methyl-2-thiopseudourea (29 mg, 1.2 mmol) was added. The reaction was stirred at rt for 12 h. The reaction mixture was washed with HCl (1M, 0.025 L), extracted with DCM (3 x 0.010 L), DCM was removed in vacuo and the product was purified by column chromatography using DCM: MeOH (25:1) to give the product 10b. (50 mg, 84%). 1_{H NMR} (400 MHz, CDCl3) δ 11.40 (s, 1H), 8.54 (d, J = 8.7 Hz, 1H), 7.39 (s, 1H), 7.28 (s, 1H), 6.85 (s, 1H), 5.89 (s, 1H), 5.32 (s, 1H), 5.20 (t, J = 9.5 Hz, 1H), 5.03 (t, J = 5.7 Hz, 1H), 4.82 (d, J = 9.4

Hz, 1H), 4.49 (d, J = 10.5 Hz, 1H), 4.37 (dd, J = 19.7, 10.0 Hz, 1H), 4.14 (dd, J = 14.2, 7.1 Hz,

1H), 4.05 (d, J = 9.2 Hz, 1H), 3.80 (s, 3H), 3.68 (dd, J = 24.9, 12.7 Hz, 1H), 3.27 (t, J = 6.8 Hz,

72 2H), 1.50 (s, 18H), 1.38 (s, 4H), 1.27 (dd, J = 7.7, 6.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 171.0, 162.8, 162.4, 162.2, 162.1, 157.1, 157.1, 152.5, 144.9, 118.2, 115.3, 110.5, 83.7, 79.7, 69.4, 68.7, 62.4, 53.5, 52.31, 51.26, 49.4, 47.1, 45.7, 41.2, 29.4, 28.7, 28.3, 28.2, 28.1, 27.9, 27.9, 26.3, 26.2, 22.7, 8.4. HRMS (ESI) Calculated for C30H50N8O12: 714.3548; Found: 715.3627 (M+H).

Compound 11a:

To a solution of 10a (54 mg, 0.088 mmol) in THF/H2O (1.0 ml, 1:1), 4 (18 mg, 0.036 mmol) was added. CuSO4 (15 mg, 0.10 mmol) was added along with sodium L-ascorbate (0.020 g, 0.10 mmol ). The reaction was stirred at rt for 12 h. Upon completion by TLC, solvent was removed in vacuo and the product was purified by column chromatography using EtOAc: MeOH (30:1) to give 11a. (48 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 8.23(s, 3H), 7.83 (s, 2H), 6.67 (s, 2H), 5.95 (s, 2H), 5.64 (s, 1H), 5.24(s,3H), 4.60(m, 6H), 4.33(s, 8H), 4.12 (s, 8H), 3.98 (s, 2H), 3.71 (s, 6H), 3.04 (d, J =33.2Hz, 4H)2.40 (s, 1H), 2.19 (s, 2H), 2.09 (s, 2H), 2.06 (s, 2H),

1.90 (s, 10H), 1.68 (s, 2H), 1.44 (s, 14H), 1.26– 1.36 (m, 42H). HRMS (ESI) Calculated for C80H122N16O25: 1706.8767; Found: 1707.8838(M+H).

73 To a solution of 10b (40 mg, 0.056 mmol) in THF/H2O (1.0 ml, 1:1), 4 (13 mg, 0.025 mmol) was added. CuSO4 (14 mg, 0.088 mmol) was added with sodium L-ascorbate (17 mg, 0.088 mmol). The reaction was stirred at rt for 12 h. Upon completion, solvent was removed in vacuo and the product was purified by column chromatography using EtOAc : MeOH (20:1) to give 9. (38 mg, 80%). 1H NMR (400 MHz, CDCl3) δ 11.38 (s, 1H), 9.74 (s, 1H), 8.19 (m, 5H), 7.28 (s, 2H), 5.89 (s, 2H), 5.31 (s, 6H), 5.21 – 5.05 (m, 2H), 4.78 (s, 2H), 4.52 (s, 5H), 4.17 – 3.90 (m, 4H),4.09-3.85 (m, 4H), 3.80-3.74 (m, 8H), 3.54 (s, 2H), 3.36 (s, 1H), 3.30 – 2.72 (m, 6H), 2.35 (d, J = 13.9 Hz, 2H), 2.05 (s, 2H), 1.81 (s, 7H), 1.54 (m, 22H), 1.45 – 1.33 (m, 12H),

1.33 – 0.99 (m, 28H), 0.90-0.84 (m, 10H). HRMS(ESI) Calculated for C86H134N20O29: 1910.9626; Found: 1912.0572(M+H)

Compound SC5:

To a solution of compound 10a (1.8 mg, 0.29 µmol) in MeOH (5.0 ml), NaOH (0.50 M, 1.0 ml) was added. The solution was stirred at rt for 2 h. The progress of the reaction was

74 monitored by TLC. Upon completion, the reaction mixture was neutralized by H+ resin, the suspension was filtered. The filtrate was collected and dried in vacuo. The residue was added to DCM/TFA (5.0 ml, 1:1), the mixture was stirred at rt for 1 h. DCM was removed in vacuo, the product was dissolved in EtOH (5.0 ml), Lindlar catalyst (10%) was added. H2 gas was bubbled to the solution and stirred at rt for 12 h. The suspension was filtered and the filtrate, which contained product SC5, was collected and crude product was purified by Biogel P2 column to give pure SC5 (1.0 mg, 76%). 1H NMR (400 MHz, D2O) δ 5.73 (s, 1H), 4.34 – 4.23 (m, 2H), 4.16 – 4.04 (m, 2H), 3.71 – 3.57 (m, 1H), 3.39 (dt, J = 13.3, 6.8 Hz, 2H), 3.02 (s, 4H), 1.98 (s,

3H), 1.55 – 1.30 (m, 4H), 1.23-1.18 (m, 4H). HRMS (ESI) Calculated for C18H32N4O8, 432.2220. Found: 433.2292 (M+H).

Compound SC6:

SC6 was synthesized in a manner similar to that of SC5 using 10b (1.6 mg, 0.22 µmol) and the crude product was purified by P2 column to give the SC6 (0.75 mg, 72%). 1H NMR (400 MHz, D2O) δ 5.63 (s, 1H), 4.38 – 4.18 (m, 2H), 4.10-3.87 (m, 2H), 3.74-3.61(m, 2H), 3.38 (t, J = 7.2 Hz, 1H), 3.02 (s, 4H), 1.93 (s, 3H), 1.52 – 0.99 (m, 8H). HRMS (ESI) Calculated for

75 Compound SC7:

To a solution of compound 11a (4.5 mg, 0.26 µmol) in MeOH (5.0 ml), NaOH (0.50 M, 1.0 ml) was added. The solution was stirred at rt for 2 h. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was neutralized by H+ resin and the suspension was filtered. The filtrate was dried in vacuo and DCM/TFA (1:1, 5.0 ml) was added to the residue and was stirred at rt for 1 h. After removal of solvent, the crude product was purified by P2 column to give SC7. (2.4 mg, 70%). 1H NMR (400 MHz, D2O) δ 7.89 (s, 2H), 7.80 (s, 3H), 5.91 (s, 2H), 4.84 (d, J = 9.4 Hz, 2H), 4.54 (s, 2H), 4.47 (d, J = 10.5 Hz, 2H), 4.29

(s, 3H), 4.25 – 4.08 (m, 3H), 3.96 (s, 4H), 3.53 (d, J = 9.8 Hz, 2H), 3.37 (dd, J = 11.9, 6.4 Hz,

2H), 2.88 (d, J = 7.4 Hz, 4H), 1.90 (s, 6H), 1.76 (s, 2H), 1.57 (s, 2H), 1.30 (s, 6H), 1.13-1.05 (m,

14H), 0.70-0.63 (m, 6H). HRMS (ESI) Calculated for C57H86N16O19: 1298.6255; Found: 1299.6329(M+H).

76 SC8 was synthesized in a manner similar to SC7 using 11b (3.5 mg, 0.18 µmol) and the crude product was purified by P2 column to give SC8. (2.0 mg, 79%). 1_{H NMR (400 MHz, D}

2O)

δ 7.89 (s, 5H), 5.80 (d, J = 17.5 Hz, 2H), 4.44 (d, J = 10.1 Hz, 2H), 4.38 (d, J = 8.5 Hz, 2H), 4.34

– 4.20 (m, 4H), 4.16 (dd, J = 26.4, 10.1 Hz, 2H), 4.01 (dt, J = 13.9, 6.9 Hz, 4H), 3.62 (d, J = 8.8

Hz, 2H), 3.51 (d, J = 11.6 Hz, 1H), 2.99 – 2.79 (m, 5H), 2.29 (d, J = 7.5 Hz, 2H), 1.96 (s, 2H),

1.95 – 1.85 (m, 5H), 1.82 (d, J = 14.9 Hz, 2H), 1.71 (s, 3H), 1.54 (s, 4H), 1.27 (s, 8H), 1.20 –

1.11 (m, 6H), 1.06 (s, 6H). HRMS (ESI) Calculated for C57H86N16O19:1382.6691; Found: 1383.6761(M+H).